US10196664B2 - Nicotiana benthamiana plants deficient in fucosyltransferase activity - Google Patents

Nicotiana benthamiana plants deficient in fucosyltransferase activity Download PDF

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US10196664B2
US10196664B2 US14/347,752 US201214347752A US10196664B2 US 10196664 B2 US10196664 B2 US 10196664B2 US 201214347752 A US201214347752 A US 201214347752A US 10196664 B2 US10196664 B2 US 10196664B2
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Koen Weterings
Gerben Van Eldik
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Icon Genetics AG
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    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
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    • C12N15/8245—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine involving modified carbohydrate or sugar alcohol metabolism, e.g. starch biosynthesis
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
    • C12N15/8245—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine involving modified carbohydrate or sugar alcohol metabolism, e.g. starch biosynthesis
    • C12N15/8246—Non-starch polysaccharides, e.g. cellulose, fructans, levans
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8257—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits for the production of primary gene products, e.g. pharmaceutical products, interferon
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1048—Glycosyltransferases (2.4)
    • C12N9/1051—Hexosyltransferases (2.4.1)
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    • C12Y204/00—Glycosyltransferases (2.4)
    • C12Y204/01—Hexosyltransferases (2.4.1)
    • C12Y204/01214—Glycoprotein 3-alpha-L-fucosyltransferase (2.4.1.214)

Definitions

  • the current invention relates to the field of molecular farming, i.e. the use of plants and plant cells as bioreactors to produce peptides and proteins, including biopharmaceuticals, particularly polypeptides and proteins with pharmaceutical interest such as therapeutic proteins, which have an altered N-glycosylation pattern resulting in a lower level of immunogenic protein-bound N-glycans, particularly a lower level of beta(1,2)-xylose residues and core alfa(1,3)-fucose residues on the protein-bound N-glycans, than counterpart unmodified plants.
  • the invention relates to plants of the genus Nicotiana which are deficient in alfa(1,3)-fucosyltransferase and beta(1,2)-xylosyltransferase activity, which plants may be applied as host plants or host cells to produce heterologous glycoproteins.
  • Glycosylation is the covalent linkage of an oligosaccharide chain to a protein resulting in a glycoprotein.
  • the oligosaccharide chain is attached to the amide nitrogen of an asparagine (Asn) residue and leads to N-glycosylation.
  • Glycosylation represents the most widespread post-translational modification found in natural and biopharmaceutical proteins. It is estimated that more than half of the human proteins are glycosylated and their function frequently depends on particular glycoforms (glycans), which can affect their plasma half life, tissue targeting or even their biological activity. Similarly, more than one-third of approved biopharmaceuticals are glycoproteins and both their function and efficiency are affected by the presence and composition of their N-glycans.
  • Leafy crops such as the tobacco plant Nicotiana benthamiana
  • Nicotiana benthamiana are an attractive system for the production of therapeutic proteins, as plants are generally considered to have several advantages, including the lack of animal pathogens such as prions and viruses, low cost and the large-scale production of safe and biologically active valuable recombinant proteins, the case of scale-up, efficient harvesting and storage possibilities.
  • N-linked glycans from plants differ from those of mammalian cells.
  • beta(1,2)-xylose and alfa(1,3)-fucose residues have been shown to be linked to the core Man3GlucNAc2-Asn of glycans, whereas they are not detected on mammalian glycans, where sialic acid residues and terminal beta(1,4)-galactosyl structures occur instead.
  • the unique N-glycans added by plants could impact both immunogenicity and functional activity of the protein and, consequently, may represent a limitation for plants to be used as a protein production platform. Indeed, the immunogenicity of beta(1,2)-xylose residues and alfa(1,3)-fucose in mammals has been described (Bardor et al., 2003, Glycobiology 13: 427).
  • beta(1,2)-xylosyltransferase The enzyme that catalyses the transfer of xylose from UDP-xylose to the core ⁇ -linked mannose of protein-bound N-glycans is beta(1,2)-xylosyltransferase (“XylT”, EC 2.4.2.38).
  • the beta-1,2-xylosyltransferase is an enzyme unique to plants and some non-vertebrate animal species and does not occur in human beings or in other vertebrates.
  • WO2007107296 describes the identification and cloning of beta-1,2-xylosyltransferases from the genus Nicotiana such as Nicotiana benthamiana.
  • the enzyme that catalyses the transfer of fucose from GDP-fucose to the core ⁇ -linked N-acetyl glucosamine (GlcNAc) of protein-bound N-glycans is alfa(1,3)-fucosyltransferase (“FucT”, EC 2.4.1.214).
  • WO2009056155 describes an alfa(1,3)-fucosyltransferase cDNA sequence from Nicotiana benthamiana.
  • WO2008141806 describes knock-outs in two alfa(1,3)-fucosyltransferase genes and in one beta(1,2)-xylosyltransferase gene in Arabidopsis thaliana .
  • WO2009056155 describes an RNA interference strategy for the generation of Nicotiana benthamiana plants which are deficient in the formation of beta-1,2-xylosyl structures as well as devoid of alfa-1,3-fucosyl structures on heterologous glycoproteins. Yin et al.
  • RNA interference strategy (2011, Protein Cell 2:41) report downregulation of the expression of the endogenous xylosyltranferase and fucosyltransferase in Nicotiana tabacum using RNA interference (RNAi) strategy. They found that xylosylated and core fucosylated N-glycans were significantly, but not completely, reduced in the glycoengineered lines.
  • WO2010145846 describes knock-outs of the two beta(1,2)-xylosyltransferase genes in Nicotiana benthamiana . The homozygous combination of the four beta(1,2)-xylosyltransferase null alleles proved to be sufficient for the elimination of the complete beta-1,2-xylosyltransferase activity in Nicotiana benthamiana.
  • the current invention provides methods and means to reduce the levels of core alfa(1,3)-fucose residues on N-glycans on glycoproteins in Nicotiana benthamiana , as will become apparent from the following description, examples, drawings and claims provided herein.
  • the invention provides a method to produce glycoproteins with reduced levels of core alfa(1,3)-fucose residues in Nicotiana benthamiana , said method comprising the steps of providing a plant or plant cell comprising at least three knock-out alfa(1,3)-fucosyltransferase genes, and cultivating said cell and isolating glycoproteins from said cell.
  • said method further comprises a reduction of the level of beta(1,2)-xylosyltransferase activity.
  • said reduction of the level of beta(1,2)-xylosyltransferase activity is the result of a knock-out mutation in endogenous beta(1,2)-fucosyltransferase genes.
  • a method is provided to produce glycoproteins with reduced levels of core alfa(1,3)-fucose residues in Nicotiana benthamiana , said method comprising the steps of providing a plant or plant cell comprising at least five knock-out alfa(1,3)-fucosyltransferase genes, and cultivating said cell and isolating glycoproteins from said cell.
  • said knock-out alfa(1,3)-fucosyltransferase genes occur in a homozygous state in the genome.
  • the methods according to the invention are further characterized in that the expression of at least five endogenous alfa(1,3)-fucosyltransferase encoding genes is reduced through transcriptional or post-transcriptional silencing.
  • the plant or plant cell according to the invention further comprises at least one chimeric gene comprising the following operably linked DNA fragments: a plant-expressible promoter, a DNA region, which when transcribed yields an RNA molecule inhibitory to at least one alfa(1,3)-fucosyltransferase encoding gene, and a DNA region comprising a transcription termination and polyadenylation signal functional in plants.
  • said DNA region comprises the sequence of SEQ ID No. 19.
  • said glycoprotein is a heterologous protein.
  • said heterologous glycoprotein is expressed from a chimeric gene comprising the following operably linked nucleic acid molecules: a plant-expressible promoter, a DNA region encoding said heterologous glycoprotein, and a DNA region involved in transcription termination and polyadenylation.
  • the method according to the invention further comprises the step of purification of said heterologous glycoprotein.
  • a glycoprotein is provided which is obtained by the methods according to the invention.
  • a glycoprotein with reduced levels of core alfa(1,3)-fucose residues is provided which is obtained by the methods according to the invention.
  • a glycoprotein with reduced levels of core alfa(1,3)-fucose and beta(1,2)-xylose residues is provided which is obtained by the methods according to the invention.
  • Nicotiana benthamiana plant or a cell, part, seed or progeny thereof, comprising at least three knock-out alfa(1,3)-fucosyltransferase genes.
  • Yet another embodiment of the invention provides a Nicotiana benthamiana plant, or a cell, part, seed or progeny thereof, comprising at least five knock-out alfa(1,3)-fucosyltransferase genes.
  • said plant or plant cell is homozygous for the knock-out alfa(1,3)-fucosyltransferase genes.
  • said plant or plant cell further comprises at least one knock-out beta(1,2)-xylosyltransferase gene, wherein said knock-out beta(1,2)-xylosyltransferase gene comprises a mutated DNA region consisting of one or more inserted, deleted or substituted nucleotides compared to a corresponding wild-type DNA region in the beta(1,2)-xylosyltransferase gene and wherein said knock-out beta(1,2)-xylosyltransferase gene does not encode a functional beta(1,2)-xylosyltransferase protein.
  • the said plant or plant cell further comprises at least one chimeric gene comprising the following operably linked DNA fragments: a plant-expressible promoter; a DNA region, which when transcribed yields an RNA molecule inhibitory to at least one alfa(1,3)-fucosyltransferase encoding gene; and a DNA region comprising a transcription termination and polyadenylation signal functional in plants.
  • said DNA region comprises the sequence of SEQ ID No. 19.
  • said plant or plant cell further comprises a glycoprotein foreign to said plant or plant cell.
  • said glycoprotein is expressed from a chimeric gene comprising the following operably linked nucleic acid molecules: a plant-expressible promoter, a DNA region encoding said heterologous glycoprotein, and a DNA region involved in transcription termination and polyadenylation.
  • knock-out alleles of alfa(1,3)-fucosyltransferase genes are provided.
  • Yet another embodiment provides the use of the methods according to the invention to obtain glycoproteins with a reduced level of core alfa(1,3)-fucose residues.
  • a further embodiment provides the use of the methods according to the invention to obtain glycoproteins with a reduced level of core alfa(1,3)-fucose residues and with a reduced level of beta(1,2)-xylose residues.
  • FIG. 1 Results from Southern blot hybridization of N. benthamiana genomic DNA hybridized with a cDNA probe of FucTA from N. benthamiana .
  • lane 1 lanes 2-7: N. benthamiana genomic DNA digested with EcoRV (lane 2), HindIII (lane 3), EcoRI (lane 4), NsiI (lane 5), AseI (lane 6), PstI (lane 7);
  • lane 8 Nicotiana tabacum cv. SR1 digested with EcoRV and HindIII.
  • FIG. 2 Example of a Southern blot comparing hybridization patterns of BAC clones (lanes 1-15) with the hybridization pattern of N. benthamiana genomic DNA (c).
  • FIG. 3 Determining optimum EMS dose for production of M2 seeds in N. benthamiana . Seeds were treated with different concentrations of EMS. A: Germination rate 6 days (black bars) and 12 days (white bars) after sowing. B: Seed survival. C: plant fertility.
  • FIG. 4 Crossing scheme used to obtain homozygous seven-fold knock out plants.
  • x14 mutant allele XYL001 (XylTg14-1 as described in WO2010145846), x19: XYL002 (XylTg19-1 as described in WO2010145846), a: FucT004, b: FucT006, c: FucT007, d: FucT009, e: FucT003.
  • the “x14/x14 x19/x19” refers to the double knock XylT mutant previously described in WO2010145846.
  • FIG. 5 Setting up and testing the complementation assay for functionality of N. benthamiana FucT genes and mutant genes.
  • WT A. thaliana wildtype
  • 3KO A. thaliana triple mutant (T-DNA-insertion knock-out mutant for XylT and FucTA and FucTB);
  • At3KO+NbFucTA triple mutant transformed with T-DNA carrying N. benthamiana FucTA cDNA
  • At3KO+mut FucTA triple mutant transformed with T-DNA carrying N. benthamiana FucTA cDNA carrying a point mutation creating a stop codon in exon 1 at position 217 of SEQ ID No. 1.
  • FIG. 6 Comparison of fucosylation levels of protein samples from N. benthamiana plants in which different FucT genes have been knocked out. Western blot analysis of leaf protein samples from plants in which different FucT genes have been knocked out. Probed with anti- ⁇ 1,3 fucose antibody (1/500 dilution); 3 min. exposure for chemoluminescence.
  • WT Wild Type plant
  • M Protein Marker. Knocked-out versions of the gene are indicated in the table as lower case; wild type version as upper case.
  • FIG. 7 Comparison of relative glycan levels on leaf proteins from N. benthamiana plants carrying null mutations for four or five FucT genes. Total protein was isolated from leaves of plants in which different FucT genes were mutated. Glycans were isolated and analyzed by MALDI-TOF. Relative levels are expressed as percentage of the total peak area as determined from the MALDI-TOF spectra.
  • White bars wild-type; Black bars: 4KO: FucTA (FucT004), -B (FucT006), -C (FucT007), and -D (FucT009) knocked out (average of three lines); Gray bars: 5KO: all FucT genes knocked out (FucT004, -006, -007, -009, and -003) (average of three lines).
  • FIG. 8 Comparison of relative glycan levels on leaf proteins from N. benthamiana plants in which all XylT and/or FucT genes have been knocked out (FucT004, -006, -007, -009, and -003, and XylTg14-1 and XylTg19-1 as described in WO2010145846).
  • Total protein was isolated from leaves of plants in which all XylT and/or FucT genes were mutated.
  • Glycans were isolated and analyzed by MALDI-TOF. Relative levels are expressed as percentage of the total peak area as determined from the MALDI-TOF spectra. White bars: wild-type.
  • RNAi plants expressing XylT and FucT RNAi genes (Strasser et al. 2008, Plant Biotech J 6:392).
  • FIG. 9 LC-MS analysis of glycans on an IgG1 expressed in a full knock-out N. benthamiana plant using magnICON®.
  • the upper panel shows a wider mass spectrum to illustrate the presence of non-glycosylated peptides.
  • Peptide 1 EQYNSTY
  • peptide 2 TKPREEQYNSTYR
  • FIG. 10 Structure of N-glycans (See also http://www.proglycan.com for a current nomenclature of N-glycans). * indicates the bond between the indicated sugar chain and an asparagine of the peptidic part of the resulting glycoprotein.
  • FIG. 11 Comparison of fucosylation levels of protein samples from N. benthamiana plants in which 6 or 7 genes have been knocked out. Plants containing the FucT RNAi gene are compared with plants which do not contain this gene. Western blot analysis of leaf protein samples. Probed with anti- ⁇ 1,3 fucose antibody (1/500 dilution); 1 hour exposure for chemoluminescence. WT: Wild Type plant; M: Protein Marker. Knocked-out versions of the gene are indicated in the table as lower case; wild type version as upper case.
  • FIG. 12 Quantitative overview of fucosylated respectively xylosylated N-glycans present on the endogenous proteins of WT, 4-, 5-, 7-fold KO, RNAi and 7KO/FucT RNAi plants.
  • Total protein was isolated from leaves of plants and glycans were isolated and analyzed by MALDI-TOF. Glycan levels are expressed as the sum of all different fucosylated respectively xylosylated N-glycan peaks as determined from the MALDI-TOF spectra.
  • WT wild-type (average of two lines).
  • RNAi plants expressing XylT and FucT RNAi genes (Strasser et al.
  • HOM7KO all FucT and XylT genes knocked out (average of three lines).
  • HET7KO+RNAi XylT and FucTA genes knocked out and other FucT genes are heterozygously knocked out combined with the FucT RNAi gene (average of four lines).
  • HOM7KO+FucT RNAi plants homozygous for all seven knock-out genes and containing the FucT RNAi gene (average of four lines).
  • the current invention is based on the identification of five genes encoding alfa(1,3)-fucosyltransferase in Nicotiana benthamiana , and that knocking-out more of these genes progressively reduces the levels of core alfa(1,3)-fucose residues on proteins produced in said plant.
  • the invention provides a method to produce glycoproteins with reduced levels of core alfa(1,3)-fucose residues in Nicotiana benthamiana , said method comprising the steps of providing a plant or plant cell comprising at least three knock-out alfa(1,3)-fucosyltransferase genes, and cultivating said cell and isolating glycoproteins from said cell.
  • control plant is generally a selected target plant which may be any plant, and may advantageously be selected among tobacco and related species like Nicotiana , including N. benthamiana, N. tabacum , and S. tuberosum , or other plants such as M. sativa .
  • the alfa(1,3)-fucosyltransferase gene is unmodified and it has wild-type levels of alfa(1,3)-fucosyltransferase activity.
  • Wild type levels of alfa(1,3)-fucosyltransferase activity refers to the typical level of alfa(1,3)-fucosyltransferase activity in a plant as it most commonly occurs in nature. Said control plant has thus not been provided either with a silencing nucleic acid molecule targeted to the endogenous alfa(1,3)-fucosyltransferase encoding gene or with an allele of an alfa(1,3)-fucosyltransferase gene associated with a low level of ⁇ -1,3-fucosyltransferase activity, such as a knock-out allele.
  • Said reduced levels of core alfa(1,3)-fucose residues can consist of a reduction of at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90%, or at least 95%, or at least 97%, or at least 99%.
  • the amount of alfa(1,3)-fucosylated glycan structures associated with a produced glycoprotein can be determined according to the methods described in this invention.
  • Core alfa(1,3)-fucose residues also “alfa(1,3)-fucose residues”, or “alpha(1,3)-fucose residues” or “ ⁇ (1,3)-fucose residues” as used herein refers to a fucose that is alpha 1,3-linked to the core region of N-glycans.
  • Alfa(1,3)-fucosyltransferase or “alpha(1,3)-fucosyltransferase”, or ⁇ (1,3)-fucosyltransferase”, or “FucT” is an enzyme that catalyses the transfer of fucose from GDP-fucose to the core ⁇ -linked N-acetyl glucosamine (GlcNAc) of protein-bound N-glycans (EC 2.4.1.214).
  • Genes encoding alfa(1,3) fucosyltransferase (FucT) in plants include the following database entries identifying experimentally demonstrated and putative FucT cDNA and gene sequences, parts thereof or homologous sequences: NM 112815 ( Arabidopsis thaliana ), NM103858 ( Arabidopsis thaliana ), AJ 618932 ( Physcomitrella patens ) At1g49710 ( Arabidopsis thaliana ), At3g19280 ( Arabidopsis thaliana ).
  • DQ789145 ( Lemna minor ), AY557602 ( Medicago truncatula ) Y18529 ( Vigna radiata ) AP004457 ( Oryza sativa ), AJ891040 encoding protein CAI70373 ( Populus alba ⁇ Populus tremula ) AY082445 encoding protein AAL99371 ( Medicago sativa ) AJ582182 encoding protein CAE46649 ( Triticum aestivum ) AJ582181 encoding protein CAE46648 ( Hordeum vulgare ), and EF562630.1 ( Nicotiana benthamiana ) (all sequences herein incorporated by reference).
  • a “Knock-out alfa(1,3)-fucosyltransferase gene” or “knock-out alfa(1,3)-fucosyltransferase allele” or “knock-out allele of the alfa(1,3)-fucosyltransferase gene” or “knock-out FucT gene” or “knock-out FucT allele” as used herein refers to a gene or an allele of said gene which does not complement the Arabidopsis thaliana triple knock-out as described by Kang et al. (2008, Proc Natl Acad Sci USA 105: 5933), using the methods as described in this invention.
  • Said “knock-out alfa(1,3)-fucosyltransferase gene” is a wild-type alfa(1,3)-fucosyltransferase gene or allele, which comprises one or more mutations in its nucleic acid sequence.
  • Said knock-out gene can, for example, be a gene that is not transcribed into a functional mRNA, or a gene of which the encoded RNA is not spliced correctly, or a gene not encoding a functional protein.
  • Knock-out genes may thus comprise, for example, genes with mutations in promoter regions, with mutations in splice-sites, or with mutations coding sequences resulting in amino acid substitutions or resulting in premature translation termination.
  • a mutation can be a deletion, an insertion or a substitution of one or more nucleotides. Mutations can be either “natural mutations” which are mutations found in nature (e.g. produced spontaneously without human application of mutagens) or “induced mutations”, which are induced by human intervention, e.g. by mutagenesis and are called non-natural mutant null alleles.
  • “Mutagenesis”, as used herein, refers to the process in which plant cells (e.g., a plurality of Nicotiana benthamiana seeds or other parts, such as pollen, etc.) are subjected to a technique which induces mutations in the DNA of the cells, such as contact with a mutagenic agent, such as a chemical substance (such as ethylmethylsulfonate (EMS), ethylnitrosourea (ENU), etc.) or ionizing radiation (neutrons (such as in fast neutron mutagenesis, etc.), alpha rays, gamma rays (such as that supplied by a Cobalt 60 source), X-rays, UV-radiation, etc.), or a combination of two or more of these.
  • a mutagenic agent such as a chemical substance (such as ethylmethylsulfonate (EMS), ethylnitrosourea (ENU), etc.) or ionizing
  • the desired mutagenesis of one or more alfa(1,3)-fucosyltransferase genes may be accomplished by use of chemical means such as by contact of one or more plant tissues with ethylmethylsulfonate (EMS), ethylnitrosourea, etc., by the use of physical means such as x-ray, etc, or by gamma radiation, such as that supplied by a Cobalt 60 source. While mutations created by irradiation are often large deletions or other gross lesions such as translocations or complex rearrangements, mutations created by chemical mutagens are often more discrete lesions such as point mutations.
  • chemical means such as by contact of one or more plant tissues with ethylmethylsulfonate (EMS), ethylnitrosourea, etc.
  • EMS alkylates guanine bases, which results in base mispairing: an alkylated guanine will pair with a thymine base, resulting primarily in G/C to A/T transitions.
  • Nicotiana benthamiana plants are regenerated from the treated cells using known techniques. For instance, the resulting Nicotiana benthamiana seeds may be planted in accordance with conventional growing procedures and following self-pollination seed is formed on the plants. Additional seed that is formed as a result of such self-pollination in the present or a subsequent generation may be harvested and screened for the presence of mutant alfa(1,3)-fucosyltransferase genes.
  • DeleteageneTM Delete-a-gene; Li et al., 2001, Plant J 27: 235-242
  • PCR polymerase chain reaction
  • TILLING targeted induced local lesions in genomes
  • Mutant alfa(1,3)-fucosyltransferase genes may be generated (for example induced by mutagenesis) and/or identified using a range of methods, which are conventional in the art, for example using PCR based methods to amplify part or all of the alfa(1,3)-fucosyltransferase genomic or cDNA and direct sequencing.
  • plants are grown from the treated seeds, or regenerated from the treated cells using known techniques.
  • mutagenized seeds may be planted in accordance with conventional growing procedures and following self-pollination seed is formed on the plants. Additional seed which is formed as a result of such self-pollination in the present or a subsequent generation may be harvested and screened for the presence of mutant alfa(1,3)-fucosyltransferase genes, using techniques which are conventional in the art, for example polymerase chain reaction (PCR) based techniques (amplification of the alfa(1,3)-fucosyltransferase genes) or hybridization based techniques, e.g.
  • PCR polymerase chain reaction
  • SNP detection methods conventional in the art can be used, for example oligo-ligation-based techniques, single base extension-based techniques, techniques based on differences in restriction sites, such as TILLING, or direct sequencing and comparing the sequences to wild-type sequeces using, for example, NovoSNP (Weckx et al, 2005, Genome Res 15: 436).
  • mutant alfa(1,3)-fucosyltransferase gene can thus be characterized by the location and the configuration of the one or more deleted, inserted, or substituted nucleotides in the wild type alfa(1,3)-fucosyltransferase gene.
  • primers and probes can be developed which specifically recognize the mutant alfa(1,3)-fucosyltransferase gene in biological samples (such as samples of plants, plant material or products comprising plant material).
  • allele(s) means any of one or more alternative forms of a gene at a particular locus.
  • alleles of a given gene are located at a specific location or locus (loci plural) on a chromosome.
  • loci plural locus
  • One allele is present on each chromosome of the pair of homologous chromosomes.
  • a method is provided to produce glycoproteins with reduced levels of core alfa(1,3)-fucose residues and reduced levels of beta(1,2)-xylose residues in Nicotiana benthamiana , said method comprising the steps of: providing a plant cell comprising at least three knock-out alpha(1,3)-fucosyltransferase genes; and having a reduced level of beta(1,2)-xylosyltransferase activity; and cultivating said cell and isolating glycoproteins from said cell.
  • “Reduced levels of beta(1,2)-xylose residues” as used herein is meant to be a reduction of levels of core beta(1,2)-xylose residues with respect to levels as obtained in control plants.
  • the “control” plant is generally a selected target plant which may be any plant and may advantageously be selected among tobacco and related species like Nicotiana , including N. benthamiana, N. tabacum , and S. tuberosum , or other plants such as M. sativa .
  • the beta(1,2)-xylosyltransferase gene is unmodified and it has wild-type levels of beta(1,2)-xylosyltransferase activity.
  • Wild type levels of beta(1,2)-xylosyltransferase activity refers to the typical level of beta(1,2)-xylosyltransferase activity in a plant as it most commonly occurs in nature. Said control plant has thus not been provided either with a silencing nucleic acid molecule targeted to the endogenous beta(1,2)-xylosyltransferase encoding gene or with an allele of an beta(1,2)-xylosyltransferase gene associated with a low level of beta(1,2)-xylosyltransferase activity, such as a knock-out allele.
  • Said reduced levels of beta(1,2)-xylosyltransferase residues can consist of a reduction of at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90%, or at least 95%, or at least 97%, or at least 99%.
  • the amount of beta(1,2)-xylosylated glycan structures associated with a produced glycoprotein can be determined according to the methods described in this invention.
  • “Reduced levels of core alfa(1,3)-fucose residues and reduced levels of beta(1,2)-xylose residues” can consist of a reduction of the levels of glycans comprising alfa(1,3)-fucose residues, beta(1,2)-xylose residues, or alfa(1,3)-fucose and beta(1,2)-xylose residues. Said reduction can consist of a reduction of at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90%, or at least 95%, or at least 97%, or at least 99%.
  • the amount of alfa(1,3)-fucosylated and beta(1,2)-xylosylated glycan structures associated with a produced glycoprotein can be determined according to the methods described in this invention.
  • the level of beta(1,2)-xylosyltransferase activity can be reduced by reducing the expression of endogenous beta(1,2)-xylosyltransferase encoding genes.
  • reducing the expression of a stated integer it is meant that transcription and/or translation and/or post-translational modification of the integer is inhibited or prevented or knocked-down or knocked-out or interrupted such that the specified integer has a reduced biological effect on a cell, tissue, organ or organism in which it would otherwise be expressed.
  • the level of expression of a particular gene may be determined by polymerase chain reaction (PCR) following reverse transcription of an mRNA template molecule.
  • PCR polymerase chain reaction
  • the expression level of a genetic sequence may be determined by northern hybridisation analysis or dot-blot hybridisation analysis or in situ hybridisation analysis or similar technique, wherein mRNA is transferred to a membrane support and hybridised to a “probe” molecule which comprises a nucleotide sequence complementary to the nucleotide sequence of the mRNA transcript encoded by the gene-of-interest, labeled with a suitable reporter molecule such as a radioactively-labelled dNTP (eg [alpha-32P] dCTP or [alpha-35S] dCTP) or biotinylated dNTP, amongst others.
  • a radioactively-labelled dNTP eg [alpha-32P] dCTP or [alpha-35S] dCTP
  • Expression of the gene-of-interest may then be determined by detecting the appearance of a signal produced by the reporter molecule bound to the hybridised probe molecule.
  • the rate of transcription of a particular gene may be determined by nuclear run-on and/or nuclear run-off experiments, wherein nuclei are isolated from a particular cell or tissue and the rate of incorporation of rNTPs into specific mRNA molecules is determined.
  • the expression of the gene-of-interest may be determined by RNase protection assay, wherein a labelled RNA probe or “riboprobe” which is complementary to the nucleotide sequence of mRNA encoded by said gene-of-interest is annealed to said mRNA for a time and under conditions sufficient for a double-stranded mRNA molecule to form, after which time the sample is subjected to digestion by RNase to remove single-stranded RNA molecules and in particular, to remove excess unhybridised riboprobe.
  • RNase RNase protection assay
  • the level of beta(1,2)-xylosyltransferase activity can conveniently be reduced or eliminated by transcriptional or post-transcriptional silencing of the expression of endogenous beta(1,2)-xylosyltransferase encoding genes.
  • a silencing RNA molecule is introduced in the plant cells targeting the endogenous beta(1,2)-xylosyltransferase encoding genes.
  • silencing RNA or “silencing RNA molecule” refers to any RNA molecule, which upon introduction into a plant cell, reduces the expression of a target gene.
  • silencing RNA may e.g. be so-called “antisense RNA”, whereby the RNA molecule comprises a sequence of at least 20 consecutive nucleotides having 95% sequence identity to the complement of the sequence of the target nucleic acid, preferably the coding sequence of the target gene.
  • antisense RNA may also be directed to regulatory sequences of target genes, including the promoter sequences and transcription termination and polyadenylation signals.
  • Silencing RNA further includes so-called “sense RNA” whereby the RNA molecule comprises a sequence of at least 20 consecutive nucleotides having 95% sequence identity to the sequence of the target nucleic acid.
  • Other silencing RNA may be “unpolyadenylated RNA” comprising at least 20 consecutive nucleotides having 95% sequence identity to the complement of the sequence of the target nucleic acid, such as described in WO01/12824 or U.S. Pat. No. 6,423,885 (both documents herein incorporated by reference).
  • silencing RNA is an RNA molecule as described in WO03/076619 (herein incorporated by reference) comprising at least 20 consecutive nucleotides having 95% sequence identity to the sequence of the target nucleic acid or the complement thereof, and further comprising a largely-double stranded region as described in WO03/076619 (including largely double stranded regions comprising a nuclear localization signal from a viroid of the Potato spindle tuber viroid-type or comprising CUG trinucleotide repeats).
  • Silencing RNA may also be double stranded RNA comprising a sense and antisense strand as herein defined, wherein the sense and antisense strand are capable of base-pairing with each other to form a double stranded RNA region (preferably the said at least 20 consecutive nucleotides of the sense and antisense RNA are complementary to each other).
  • the sense and antisense region may also be present within one RNA molecule such that a hairpin RNA (hpRNA) can be formed when the sense and antisense region form a double stranded RNA region.
  • hpRNA hairpin RNA
  • the hpRNA may be classified as long hpRNA, having long, sense and antisense regions which can be largely complementary, but need not be entirely complementary (typically larger than about 200 bp, ranging between 200-1000 bp). hpRNA can also be rather small ranging in size from about 30 to about 42 bp, but not much longer than 94 bp (see WO04/073390, herein incorporated by reference). Silencing RNA may also be artificial micro-RNA molecules as described e.g. in WO2005/052170, WO2005/047505 or US 2005/0144667, or ta-siRNAs as described in WO2006/074400 (all documents incorporated herein by reference).
  • a suitable method for silencing the beta(1,2)-xylosyltransferase is the method as described in WO2009056155.
  • the reduced level of beta(1,2)-xylosyltransferase is activity is the result of a knock-out mutation in endogenous beta(1,2)-xylosyltransferase genes.
  • a knock-out mutation in endogenous beta(1,2)-xylosyltransferase genes is a mutation that renders the beta(1,2)-xylosyltransferase gene inactive, wherein the inactive gene is characterized in that the gene does not encode a functional alfa(1,3)-fucosyltransferase protein.
  • Said gene also referred to as “knock-out gene” or “knock-out allele” can either be a gene that is not transcribed into a functional mRNA, or a gene of which the encoded RNA is not spliced correctly, or a gene not encoding a functional protein.
  • Mutations that render the beta(1,2)-xylosyltransferase gene inactive thus comprise, for example, mutations in the promoter regions, mutations in the splice-sites, or mutations in the coding sequences resulting in amino acid substitutions or premature translation termination.
  • Suitable knock-out mutations in endogenous beta(1,2)-xylosyltransferase genes of Nicotiana benthamiana are the knock-outs as described in WO2010145846.
  • the alfa(1,3)-fucosyltransferase and the beta(1,2)-xylosyltransferase activity can be evaluated by determining the level of alfa(1,3)-fucose and the level of beta(1,2)-xylose residues on protein-bound N-glycans from a plant, respectively.
  • the level of alfa(1,3)-fucose and the level of beta(1,2)-xylose residues on protein-bound N-glycans from a plant can be measured e.g. by Western blot analysis using fucose- or xylose specific antibodies, as described e.g. by Faye et al.
  • said plant or plant cell comprises at least five knock-out alfa(1,3)-fucosyltransferase genes.
  • At least five knock-out alfa(1,3)-fucosyltransferase genes can be five knock-out alfa(1,3)-fucosyltransferase genes, or six alfa(1,3)-fucosyltransferase genes, or seven alfa(1,3)-fucosyltransferase genes, or more than seven alfa(1,3)-fucosyltransferase genes.
  • Suitable knock-out alfa(1,3)-fucosyltransferase genes can be mutated versions of the native alfa(1,3)-fucosyltransferase genes selected from the group consisting of nucleic acids encoding the amino acid sequence of SEQ ID No. 3, SEQ ID No. 6, SEQ ID No. 9, SEQ ID No. 12, SEQ ID No. 14, or of nucleic acids encoding amino acid sequences having at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 97%, or at least 98%, or at least 99% identity to these amino acid sequences.
  • Suitable knock-out alfa(1,3)-fucosyltransferase genes can further be mutated versions of the native alfa(1,3)-fucosyltransferase genes selected from the group consisting of SEQ ID No. 1, SEQ ID No. 4, SEQ ID No. 7, SEQ ID No. 10, SEQ ID No. 13, or of nucleic acids having at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 97%, or at least 98%, or at least 99% identity to these sequences.
  • said knock-out alfa(1,3)-fucosyltransferase genes are mutated versions of the native alfa(1,3)-fucosyltransferase genes selected from the group consisting of:
  • said knock-out alfa(1,3)-fucosyltransferase genes are mutated versions of the native alfa(1,3)-fucosyltransferase genes selected from the group consisting of:
  • Suitable knock-out alfa(1,3)-fucosyltransferase genes for the invention are genes with one or more mutations selected from the group of mutations as depicted in Table 2 and Table 4.
  • said knock-out alfa(1,3)-fucosyltransferase gene is selected from the group consisting of:
  • a “mutated version” of a gene as used herein is a version of a gene which contains one or more mutations.
  • a “native alfa(1,3)-fucosyltransferase”, also “wild-type alfa(1,3)-fucosyltransferase” as used herein refers to a typical form of an alfa(1,3)-fucosyltransferase gene as it most commonly occurs in nature.
  • said knock-out alfa(1,3)-fucosyltransferase genes occur in a homozygous state in the genome.
  • the method according to the invention is further characterized in that the expression of at least five endogenous alfa(1,3)-fucosyltransferase encoding genes is reduced through transcriptional or post-transcriptional silencing.
  • Transcriptional and post-transcriptional silencing can suitably be achieved by introducing a silencing RNA molecule in the plant cells targeting the endogenous alfa(1,3)-fucosyltransferase encoding genes.
  • each of the chimeric genes encodes a silencing RNA molecule, each of which is suitable to silence at least one of the alfa(1,3)-fucosyltransferase genes.
  • one chimeric gene can be introduced in the plant cells which encodes a silencing RNA molecule capable of silencing at least five alfa(1,3)-fucosyltransferase genes.
  • Said one chimeric gene can comprise several regions of 21 consecutive nucleotides, each of which having at least 85% sequence identity to a region of 21 nucleotides occurring in at least one of the alfa(1,3)-fucosyltransferase genes.
  • said one chimeric gene can comprise a region of 21 consecutive nucleotides characterized that at least five alfa(1,3)-fucosyltransferase genes comprise a sequence of 21 nucleotides having 85% identity to said region of 21 consecutive nucleotides.
  • a suitable methods for silencing the alfa(1,3)-fucosyltransferase genes of Nicotiana benthamiana are the methods as described in WO2009056155.
  • the plant cell according to the invention comprises at least one chimeric gene comprising the following operably linked DNA fragments: a plant-expressible promoter, a DNA region, which when transcribed yields an RNA molecule inhibitory to at least one alfa(1,3)-fucosyltransferase encoding gene, a DNA region comprising a transcription termination and polyadenylation signal functional in plants.
  • said DNA region yields an RNA molecule capable of forming a double-stranded RNA region at least between an RNA region transcribed from a first sense DNA region comprising a nucleotide sequence of at least 18 out of 21 nucleotides selected from SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 11, SEQ ID NO: 13, or the complement thereof, and an RNA region transcribed from a second antisense DNA region comprising a nucleotide sequence of at least 18 consecutive nucleotides which have at least 95% sequence identity to the complement of said first sense DNA region.
  • RNA molecule inhibitory to at least one alfa(1,3)-fucosyltransferase encoding gene refers to a silencing RNA molecule which reduces the expression of at least one alfa(1,3)-fucosyltransferase encoding gene.
  • plant-expressible promoter means a DNA sequence that is capable of controlling (initiating) transcription in a plant cell. This includes any promoter of plant origin, but also any promoter of non-plant origin which is capable of directing transcription in a plant cell, i.e., certain promoters of viral or bacterial origin such as the CaMV35S (Harpster et al. (1988) Mol Gen Genet.
  • the subterranean clover virus promoter No 4 or No 7 (WO9606932), or T-DNA gene promoters but also tissue-specific or organ-specific promoters including but not limited to seed-specific promoters (e.g., WO89/03887), organ-primordia specific promoters (An et al. (1996) Plant Cell 8(1):15-30), stem-specific promoters (Keller et al., (1988) EMBO J. 7(12): 3625-3633), leaf specific promoters (Hudspeth et al. (1989) Plant Mol Biol.
  • mesophyl-specific promoters such as the light-inducible Rubisco promoters
  • root-specific promoters such as the light-inducible Rubisco promoters
  • tuber-specific promoters such as the tuber-specific promoters
  • vascular tissue specific promoters such as the vascular tissue specific promoters (Peleman et al. (1989) Gene 84: 359-369)
  • stamen-selective promoters WO 89/10396, WO 92/13956
  • dehiscence zone specific promoters WO 97/13865
  • a “transcription termination and polyadenylation region” as used herein is a sequence that drives the cleavage of the nascent RNA, whereafter a poly(A) tail is added at the resulting RNA 3′ end, functional in plants.
  • Transcription termination and polyadenylation signals functional in plants include, but are not limited to, 3′nos, 3′35S, 3′his and 3′g7.
  • the plant cell according to the invention comprises a chimeric gene comprising a plant-expressible promoter, a DNA region, which when transcribed yields an RNA molecule inhibitory to at least one alfa(1,3)-fucosyltransferase encoding gene, and a DNA region comprising a transcription termination and polyadenylation signal functional in plants, characterized in that said DNA region comprises the sequence of SEQ ID No. 19.
  • the glycoproteins produced according to the methods of the invention are heterologous glycoproteins.
  • said heterologous proteins are expressed from a chimeric gene comprising the following operably linked nucleic acid molecules: a plant-expressible promoter, a DNA region encoding said heterologous glycoprotein, a DNA region involved in transcription termination and polyadenylation.
  • the methods according to the invention further comprise the step of purification of said heterologous proteins.
  • expression shall be taken in its widest context to refer to the transcription of a particular genetic sequence to produce sense or antisense mRNA or the translation of a sense mRNA molecule to produce a peptide, polypeptide, oligopeptide, protein or enzyme molecule.
  • expression comprising the production of a sense mRNA transcript, the word “expression” may also be construed to indicate the combination of transcription and translation processes, with or without subsequent post-translational events which modify the biological activity, cellular or sub-cellular localization, turnover or steady-state level of the peptide, polypeptide, oligopeptide, protein or enzyme molecule.
  • Heterologous glycoproteins i.e. glycoproteins which are not normally expressed in such plant cells in nature, may include mammalian or human proteins, which can be used as therapeutics such as e.g. monoclonal antibodies.
  • the foreign glycoproteins may be expressed from chimeric genes comprising a plant-expressible promoter and the coding region of the glycoprotein of interest, whereby the chimeric gene is stably integrated in the genome of the plant cell. Methods to express foreign proteins in plant cells are well known in the art.
  • the foreign glycoproteins may also be expressed in a transient manner, e.g.
  • heterologous protein it is understood a protein (i.e. a polypeptide) that is not expressed by the plant or plant cells in nature. This is in contrast with a homologous protein which is a protein naturally expressed by a plant or plant cell. Heterologous and homologous polypeptides that undergo post-translational N-glycosylation are referred to herein as heterologous or homologous glycoproteins.
  • heterologous proteins of interest that can be advantageously produced by the methods of this invention include, without limitation, cytokines, cytokine receptors, growth factors (e.g. EGF, HER-2, FGF-alpha, FGF-beta, TGF-alpha, TGF-beta, PDGF, IGF-I, IGF-2, NGF), growth factor receptors.
  • growth factors e.g. EGF, HER-2, FGF-alpha, FGF-beta, TGF-alpha, TGF-beta, PDGF, IGF-I, IGF-2, NGF
  • growth factor receptors e.g. human growth hormone, bovine growth hormone
  • insulin e.g., insulin A chain and insulin B chain
  • pro-insulin erythropoietin (EPO)
  • colony stimulating factors e.g.
  • G-CSF G-CSF, GM-CSF, M-CSF
  • interleukins vascular endothelial growth factor (VEGF) and its receptor (VEGF-R), interferons, tumor necrosis factor and its receptors, thrombopoietin (TPO), thrombin, brain natriuretic peptide (BNP); clotting factors (e.g.
  • TPA tissue plasminogen activator
  • FSH follicle stimulating hormone
  • LH luteinizing hormone
  • CD proteins e.g., CD2, CD3, CD4, CD5, CD7, CD8, CDI Ia, CDI Ib, CD18, CD19, CD20, CD25, CD33, CD44, CD45, CD71, etc.
  • CTLA proteins e.g.CTLA4
  • BNPs bone morphogenic proteins
  • BNPs bone morphogenic proteins
  • BDNF bone derived neurotrophic factor
  • neurotrophins e.g. rennin, rheumatoid factor, RANTES, albumin, relaxin
  • macrophage inhibitory protein e.g. MIP-I, MIP-2
  • viral proteins or antigens e.g. MIP-I, MIP-2
  • surface membrane proteins e.g. ion channel proteins, enzymes, regulatory proteins, immunomodulatory proteins, (e.g. HLA, MHC, the B7 family), homing receptors, transport proteins, superoxide dismutase (SOD), G-protein coupled receptor proteins (GPCRs), neuromodulatory proteins, Alzheimer's Disease associated proteins and peptides.
  • SOD superoxide dismutase
  • GPCRs G-protein coupled receptor proteins
  • Fusion proteins and polypeptides, chimeric proteins and polypeptides, as well as fragments or portions, or mutants, variants, or analogs of any of the aforementioned proteins and polypeptides are also included among the suitable proteins, polypeptides and peptides that can be produced by the methods of the present invention.
  • the protein of interest can be a glycoprotein.
  • One class of glycoproteins are viral glycoproteins, in particular subunits, than can be used to produce for example a vaccine.
  • viral proteins comprise proteins from rhinovirus, poliomyelitis virus, herpes virus, bovine herpes virus, influenza virus, newcastle disease virus, respiratory syncitio virus, measles virus, retrovirus, such as human immunodeficiency virus or a parvovirus or a papovavirus, rotavirus or a coronavirus, such as transmissable gastroenteritisvirus or a flavivirus, such as tick-borne encephalitis virus or yellow fever virus, a togavirus, such as rubella virus or eastern-, western-, or venezuelean equine encephalomyelitis virus, a hepatitis causing virus, such as hepatitis A or hepatitis B virus, a pestivirus, such as hog cholera virus or a rhabdovirus, such as rabies virus.
  • retrovirus such as human immunodeficiency virus or a parvovirus or a papovavirus
  • the heterologous glycoprotein can be an antibody or a fragment thereof.
  • antibody refers to recombinant antibodies (for example of the classes IgD, IgG, IgA, IgM, IgE) and recombinant antibodies such as single-chain antibodies, chimeric and humanized antibodies and multi-specific antibodies.
  • antibody also refers to fragments and derivatives of all of the foregoing, and may further comprise any modified or derivatised variants thereof that retain the ability to specifically bind an epitope.
  • Antibody derivatives may comprise a protein or chemical moiety conjugated to an antibody.
  • a monoclonal antibody is capable of selectively binding to a target antigen or epitope.
  • Antibodies include, monoclonal antibodies (mAbs), humanized or chimeric antibodies, camelized antibodies, camelid antibodies (Nanobodies®), single chain antibodies (scFvs), Fab fragments, F(ab′) 2 fragments, disulfide-linked Fvs (sdFv) fragments, anti-idiotypic (anti-Id) antibodies, intra-bodies, synthetic antibodies, and epitope-binding fragments of any of the above.
  • the term “antibody” also refers to fusion protein that includes a region equivalent to the Fc region of an immunoglobulin. Also envisaged is the production in the plant or plant cells of the invention of so called dual-specificity antibodies (Bostrom J et al (2009) Science 323, 1610-1614).
  • Antibodies within the scope of the present invention include those comprising the amino acid sequences of the following antibodies: anti-HER2 antibodies including antibodies comprising the heavy and light chain variable regions (see U.S. Pat. No. 5,725,856) or Trastuzumab such as HERCEPTINTM; anti-CD20 antibodies such as chimeric anti-CD20 as in U.S. Pat. No. 5,736,137, a chimeric or humanized variant of the 2H7 antibody as in U.S. Pat. No.
  • anti-VEGF antibodies including humanized and/or affinity matured anti-VEGF antibodies such as the humanized anti-VEGF antibody huA4.6.1 AVASTINTM (WO 96/30046 and WO 98/45331); anti-EGFR (chimerized or humanized antibody as in WO 96/40210); anti-CD3 antibodies such as OKT3 (U.S. Pat. No. 4,515,893); anti-CD25 or anti-tac antibodies such as CHI-621 (SIMULECT) and (ZENAPAX) (U.S. Pat. No. 5,693,762).
  • the present invention provides a method for the production of an antibody which comprises culturing a transformed plant cell or growing a transformed plant of the present invention.
  • the produced antibody may be purified and formulated in accordance with standard procedures.
  • the DNA region encoding the heterologous glycoproteins may be codon optimized to increase the level of expression within the plant.
  • codon optimization it is meant the selection of appropriate DNA nucleotides for the synthesis of oligonucleotide building blocks, and their subsequent enzymatic assembly, of a structural gene or fragment thereof in order to approach codon usage in plants.
  • Purification is to isolate the heterologous protein from the mixture of total plant proteins.
  • the level of purification can be to at least 50% purity, or to at least 60% purity, or to at least 70% purity, or to at least 80% purity, or to at least 85% purity, or to at least 90% purity, or to at least 95% purity, or to at least 98% purity, or to at least 99% purity.
  • Methods for protein purification are well-known in the art and may consist of, but are not limited to, differential precipitation, ultracentrifugation, chromatography, or affinity purification.
  • glycoprotein obtained by the methods according to the invention.
  • said glycoprotein has reduced levels of alfa(1,3)-fucose residues.
  • said glycoprotein has reduced levels of alfa(1,3)-fucose residues and reduced levels of beta(1,2)-xylose residues.
  • Nicotiana benthamiana plant or a cell, part, seed or progeny thereof, comprising at least three knock-out alfa(1,3)-fucosyltransferase genes.
  • said plant comprises at least five knock-out alfa(1,3)-fucosyltransferase genes.
  • At least five knock-out alfa(1,3)-fucosyltransferase genes can be five knock-out alfa(1,3)-fucosyltransferase genes, or six knock-out alfa(1,3)-fucosyltransferase genes, or seven knock-out alfa(1,3)-fucosyltransferase genes, or at least seven knock-out alfa(1,3)-fucosyltransferase genes.
  • Suitable knock-out alfa(1,3)-fucosyltransferase genes can be mutated versions of the native alfa(1,3)-fucosyltransferase genes selected from the group consisting of nucleic acids encoding the amino acid sequence of SEQ ID No. 3, SEQ ID No. 6, SEQ ID No. 9, SEQ ID No. 12, SEQ ID No. 14, or of nucleic acids encoding amino acid sequences having at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 97%, or at least 98%, or at least 99% identity to these amino acid sequences.
  • Suitable knock-out alfa(1,3)-fucosyltransferase genes can further be mutated versions of the native alfa(1,3)-fucosyltransferase genes selected from the group consisting of SEQ ID No. 1, SEQ ID No. 4, SEQ ID No. 7, SEQ ID No. 10, SEQ ID No. 13, or of nucleic acids having at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 97%, or at least 98%, or at least 99% identity to these sequences.
  • Another embodiment provides plants according to invention, wherein one or more of the knock-out alfa(1,3)-fucosyltransferase genes is a mutated version of the native alfa(1,3)-fucosyltransferase gene selected from the group consisting of:
  • Yet another embodiment provides plants according to the invention, wherein one or more of the knock-out alfa(1,3)-fucosyltransferase genes is a mutated version of the native alfa(1,3)-fucosyltransferase gene selected from the group consisting of:
  • Yet another embodiment provides plants according to the invention wherein the knock-out alfa(1,3)-fucosyltransferase gene is selected from the group consisting of:
  • the plant or plant cell according to the invention is homozygous for the knock-out alfa(1,3)-fucosyltransferase genes.
  • the plant or plant cell according to the invention further comprises at least one knock-out beta(1,2)-xylosyltransferase gene, wherein said knock-out beta(1,2)-xylosyltransferase gene comprises a mutated DNA region consisting of one or more inserted, deleted or substituted nucleotides compared to a corresponding wild-type DNA region in the beta(1,2)-xylosyltransferase gene and wherein said knock-out beta(1,2)-xylosyltransferase gene does not encode a functional beta(1,2)-xylosyltransferase protein.
  • the said plant or plant cell further comprises at least one chimeric gene comprising the following operably linked DNA fragments: a plant-expressible promoter; a DNA region, which when transcribed yields an RNA molecule inhibitory to at least one alfa(1,3)-fucosyltransferase encoding gene; and a DNA region comprising a transcription termination and polyadenylation signal functional in plants.
  • said DNA region yields an RNA molecule capable of forming a double-stranded RNA region at least between an RNA region transcribed from a first sense DNA region comprising a nucleotide sequence of at least 18 out of 21 nucleotides selected from SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 11, SEQ ID NO: 13, or the complement thereof, and an RNA region transcribed from a second antisense DNA region comprising a nucleotide sequence of at least 18 consecutive nucleotides which have at least 95% sequence identity to the complement of said first sense DNA region.
  • said DNA region comprises the sequence of SEQ ID No. 19.
  • the plant or plant cell according to the invention further comprises a glycoprotein foreign to said plant or plant cell.
  • said glycoprotein is expressed from a chimeric gene comprising the following operably linked nucleic acid molecules: a plant-expressible promoter, a DNA region encoding said heterologous glycoprotein, a DNA region involved in transcription termination and polyadenylation.
  • Another embodiment according to the invention provides a knock-out allele of an alfa(1,3)-fucosyltransferase gene selected from the group consisting of:
  • Yet another embodiment provides the use of the methods according to the invention to obtain glycoproteins with a reduced level of core alfa(1,3)-fucose residues.
  • a further embodiment provides the use of the methods according to the invention to obtain glycoproteins with a reduced level of core alfa(1,3)-fucose residues and with a reduced level of beta(1,2)-xylose residues.
  • Plants according to the invention can be further crossed by traditional breeding techniques and can be used to produce seeds to obtain progeny plants comprising glycoproteins with reduced levels of alfa(1,3)-fucosylation and/or reduced levels of beta(1,2)-xylosylation.
  • nucleic acid or protein comprising a sequence of nucleotides or amino acids
  • a chimeric gene comprising a DNA region which is functionally or structurally defined, may comprise additional DNA regions etc.
  • FT /vntifkey “15”
  • FT /label intron ⁇ 2
  • FT /note “ Arabidopsis XylT gene intron 2”
  • FT /vntifkey “21”
  • FT /vntifkey “21”
  • FucTA and FucTB form one group
  • FucTA has 100% identity to the previously published N. benthamiana FucTA cDNA (Strasser et al. (2008) Plant Biotech J. 6:392).
  • the coding regions of FucTA and -B have 96% identity.
  • the main striking difference between the two genes is that FucTB has a shorter coding sequence due to a premature stop codon.
  • FucTC, FucTD and FucTE form the second group. All three genes have 96% identity in the coding regions. Genes from the two groups share 80% relative identity.
  • Ethyl MethaneSulfonate causes G ⁇ A and C ⁇ T point mutations by alkylating Guanine (G). These point mutations can knock out genes if they generate null mutations by inducing stop codons or splice site mutations. Using this method we can screen for knock outs for all FucT genes. A total knock out will be achieved after crossing these mutants.
  • the optimum dose for EMS mutagenesis was determined by treating seeds with 0, 50, 75, 100, 150, and 200 mM EMS. Briefly, seeds were imbibed for 2 hours at room temperature, treated with EMS for 4 hours at room temperature and washed 5 times for 15 minutes at room temperature. Seeds were dried overnight and sown immediately. The effects on germination, seedling lethality and plant fertility were recorded. As N. benthamiana most probably is an amphidiploid species from a combination of N. debneyi and N. suaveolens (Goodspeed, T. H. 1954 Pages 485-487 in: The Genus Nicotiana : Origins, Relationships and Evolution of its Species in the Light of Their Distribution, Morphology and Cytogenetics.
  • SNPs Single Nucleotide Polymorphisms
  • FucT genes the following number of EMS lines were screened: 4275 M2 individuals were screened for mutations in FucTA, 8075 for FucTB, 6555 for FucTC, 6270 for FucTD and 4370 for FucTE. The following number of putative null alleles were identified: three in FucTA, two splice site mutations and one stop codon mutation, respectively labeled FucT001, FucT004, and FucT013. Two putative null alleles, respectively one splice site mutation and one stop codon mutation, were identified for FucTB, labeled FucT006 and FucT008.
  • FucTC 4 putative null alleles were identified, respectively 1 splice site mutation and three stop codon postitions, labeled FucT007, FucT010, FucT011 and FucT012.
  • FucTD one splice site mutation and one stop codon mutation, were identified, labeled FucT005 and FucT009.
  • FucTE no stop codon mutations were identified. Instead, two alleles with substitution mutations were identified, labeled FucT002 and FucT003. The FucT003 substitution was located in the conserved “MOTIFII”.
  • Table 4 summarizes the results of the screening for FucT genes: mutation position, mutation sequence and mutant type.
  • the homozygous mutants that were selected this way were allowed to self-fertilize to create a stable mutant seedlot.
  • a selected number of mutants were entered into a 5-fold backcrossing scheme with the “BENTHAMIANA” accession to eliminate most if not all of the mutation drag.
  • a selected number of mutants were entered in a crossing scheme to produce the 7-fold knock out plants. The crossing scheme is shown in FIG. 4 .
  • the final set of mutants that were used to generate the 7-fold knock out plant was: XYL001 (XylTg14-1 as described in WO2010145846), XYL002 (XylTg19-1 as described in WO2010145846), FucT003, FucT004, FucT006, FucT007, FucT009.
  • the selection of the final set of FucT mutants was based on a gene transcription- and a complementation assay. Both are described below.
  • TaqMan probes are oligonucleotides that have a fluorescent reporter dye attached to the 5′ end and a quencher moiety coupled to the 3′ end. These probes are designed to hybridize to an internal region of a PCR product. In the unhybridized state, the proximity of the fluorescent and the quench molecules prevents the detection of a fluorescent signal from the probe.
  • cDNA was prepared from mRNA extracted from N. benthamiana leaves, following the protocol of the superscript II (Invitrogen) kit.
  • PC1 using a standard PCR mix [10 ⁇ l Go Taq buffer 5 ⁇ ; 1 ⁇ l dNTM 10 mM; 1 ⁇ l forward primer 10 ⁇ M; 1 ⁇ l reverse primer 10 ⁇ M; 0.4 ⁇ l Taq polymerase 5 U/ ⁇ l; 2 ⁇ l purified PCR product in 50 ⁇ l total volume] and standard protocol [2 min 94° C.; 30 ⁇ [30 sec 94° C., 30 sec 56° C./62° C., 30 sec 72° C.], 10 min 72° C.].
  • the resulting PCR products were purified with the Qiagen PCR purification kit, cloned in the PGemT Easy vector (Promega) and transformed into commercial thermo competent TOP10 cells (Invitrogen). 100 ⁇ l was plated out on LB plates containing 100 ⁇ g/ml triacelline. 192 clones resulting from primer combination PC1 and 96 from PC2 were sequenced by AGOWA. Based on SNPs in the five FucT sequences, it was possible to distinguish which of the different FucT genes was expressed.
  • FucTE appeared not to be expressed in N. benthamiana leaves, we decided to keep the FucTE gene as last one to cross into to the crossing scheme for the 7-fold knock out plant (see “generation 4” in FIG. 4 ).
  • the mutant to be complemented was an Arabidopsis thaliana line in which the FucT and XylT genes were knocked out by T-DNA insertion (“triple knock-out mutant”). This line has been described by Kang et al. (2008) Proc Natl Acad Sci USA and was also created in our laboratory by crossing three different T-DNA knock out lines available from SALK (see also WO2010121818).
  • FIG. 5 shows the results of a complementation assay with a FucTA in which an EMS mutation was simulated at the 8th possible stop codon (position 217; see table 3 FucTA gene). From the absence of a chemoluminescence signal in lanes 1 to 5 in the section labeled “At3KO+mut FucTA (stop in Exon1)”, it is clear that this mutated version of FucTA cannot complement the triple knock-out mutant.
  • FIG. 6 clearly shows that knocking out more FucT genes progressively removes core ⁇ 1,3 Fucosyltransferase activity from the mutant plants as indicated by the decreasing chemoluminescence signal from the bound anti- ⁇ 1,3 fucose antibody. This result indicates that probably the FucTB and -E genes still have some fucosyltransferase activity although this was not detected (i.e. compare lanes “aBcdE” versus “abcdE” and compare lanes “abcdE” versus “abcde”).
  • IgG1 was isolated from leaf extract nine days after infiltration using protein G.
  • the heavy chain of the purified antibody was isolated by cutting the corresponding band from a reducing SDS-PAGE.
  • the heavy chain protein in this band was used for glycan analysis by LC-MS as described by Kolarich et al. 2006, Proteomics 6:3369.
  • FIG. 9 shows the resulting spectrum from this analysis.
  • the upper panel shows a wider mass spectrum to illustrate the presence of non-glycosylated peptides.
  • Peptide 1 (EEQYNSTY) (SEQ ID NO:22) and peptide 2 (TKPREEQYNSTYR) (SEQ ID NO:23) are two variants from the same trypsin digestion. They differ in length caused by steric hindrance of the trypsin by the presence of N-glycans. As a result, all peptide-glycans produce two peaks in this LC-MS spectrum: indicated on the lower panel in black for glycopeptide 1 and orange for glycopeptide 2. In the lower panel of FIG.
  • Results from the Western blot analysis in FIG. 11 clearly show that adding the FucT RNAi gene to the seven-fold knock out plants further removes core ⁇ 1,3 Fucose residues from the N-glycans as indicated by the complete absence of chemoluminescence signal from the lanes containing proteins from the 7KO/FucT RNAi plants as compared to lanes containing proteins from plants in which 6 or 7 genes have been knocked out. Even after a prolonged exposure of 1 hour, no signal could be detected in 7KO/FucT RNAi lanes.
  • FIG. 12 shows a quantitative overview of fucosylated resp. xylosylated N-glycans present on the endogenous proteins of WT, 4-, 5-, 7-fold KO, RNAi and 7KO/FucT RNAi plants.
  • RNAi genes are constructed that target silencing of all FucT genes by including multiple stretches of 25 or more nucleotides that are 100% homologous to two or more FucT genes and, combined, target all FucT genes.
  • a fragment of the FucTB coding sequence (Seq ID No 5) from nucleotide 1183 to 1265 contains a stretch of 44 nucleotides, from 1183 to 1226, that is 100% homologous to FucT-B, -C, -D, and -E and a fragment of 47 nucleotides, from 1219 to 1265, that is 100% homologous to FucT-A, and -B.
  • This fragment (Seq ID No 20) is assembled into an RNAi gene as shown in Seq ID No 21. Expression of the RNAi gene is driven by the 35S promoter by cloning it into a T-DNA vector similar to pGAX3 (WO 2009/056155).
  • the seven-fold knock-out N. benthamiana plants are transformed with this construct and analyzed for N-glycan composition on endogenous proteins and on heterologously magnICON®-expressed proteins like, for instance, an IgG1 molecule.
  • the FucT RNAi gene is cloned in a promoterless T-DNA vector similar to pICH3781 and pICH3831 (WO 02/101060) where the existing BAR gene is replaced by the FucT RNAi gene fragment.
  • the seven-fold knock-out N. benthamiana plants are transformed with these constructs.
  • Use of promoterless vectors will provide a broader choice of primary transformants in comparison to vectors with strong constitutive promoter. In such case the RNAi becomes part of a transcriptional fusion with a residential gene (the promoterless vector contains splice acceptor sites in front of the RNAi gene).
  • RNAi usually targets multigene family and this might compromise plant phenotype—growth, development, abiotic or biotic stress resistance, etc.
  • the resulting stably transformed plants are screened for absence of fucoses on the N-glycans of their endogenous proteins and of heterologously magniCON®-expressed proteins like, for instance, an IgG1 molecule. Those selected can be additionally screened for their performance in glasshouses, e.g. vegetative growth efficiency in comparison with wild type plants.

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